PROJECT SUMMARY Gorham-Stout disease (GSD) and generalized lymphatic anomaly (GLA) are related diseases of the lymphatic system. GSD and GLA patients develop ectopic lymphatics in bone and gradually lose bone. Unfortunately, our understanding of the biology of GLA and GSD has not significantly advanced over the past 60 years. This gap in knowledge has made it difficult to treat GLA and GSD patients. Our long-term goal is to identify effective therapies and biomarkers for GLA and GSD. The objective of this application, which is the first step in pursuit of that goal, is to use novel mouse models developed in my laboratory to determine how lymphatic vessels form in bone and promote bone loss. PIK3CA encodes the catalytic subunit of phosphatidylinositol 3-kinase (PI3K). Our preliminary studies have revealed that mice that express an active form of PIK3CA (PIK3CALEC) in their lymphatic endothelial cells (LECs) develop irregular lymphatics in their soft tissues and bones. VEGF-C is a growth factor that stimulates PI3K signaling in LECs by activating VEGFR3. To induce excessive PI3K signaling specifically in LECs around bone, we created transgenic mice that overexpress VEGF-C in bone (VegfcOE). We have found that overexpression of VEGF-C in bone causes lymphatics to form in bone and bone loss. We have also found that rapamycin (FDA-approved mTOR inhibitor) inhibits lymphangiogenesis in PIK3CALEC mice and VegfcOE mice. This proposal will test the hypothesis that excessive PI3K/mTOR signaling in LECs stimulates the formation of lymphatic vessels in bone and osteoclast-mediated bone resorption. The specific aims of this proposal are to: 1) Characterize the development of bone lymphatics in PIK3CALEC and VegfcOE mice; 2) Determine the mechanism of bone loss in PIK3CALEC and VegfcOE mice; and 3) Determine the effect of rapamycin on lymphangiogenesis and osteolysis in PIK3CALEC and VegfcOE mice. The results from these experiments will have a significant impact on the field because they will answer fundamental questions regarding the biology of bone lymphatics. Our experiments will reveal how lymphatics form in bone and promote bone loss. This knowledge will significantly advance our understanding of processes that are relevant to the pathogenesis of GLA and GSD and could be used to inhibit lymphangiogenesis in GLA, GSD, and other human diseases.